WO1990003791A1

WO1990003791A1 - Methods for interfering with hiv multiplication and composition for the treatment of aids

Info

Publication number: WO1990003791A1
Application number: PCT/US1989/004249
Authority: WO
Inventors: Nicola Diferrante
Original assignee: Nicola Diferrante
Priority date: 1988-10-05
Filing date: 1989-09-27
Publication date: 1990-04-19
Also published as: AU4426289A

Abstract

Heparin and low molecular weight derivatives of heparin lacking anticoagulant activity are effective in interfering with the life cycle of HIV. Cells exposed to heparin or its low molecular weight derivatives one hour before or after challenge with HIV showed no evidence of viral infection when examined four to six days post HIV challenge. Control cells similarly challenged with HIV showed 100 % HIV infection six days post HIV challenge. Cells challenged with HIV as much as 48 hours before exposure to heparin or its low molecular derivatives showed little evidence of HIV multiplication and evidenced healthy morphological features.

Description

METHODS FOR INTERFERING WITH HIV MULTIPLICATION AND COMPOSITION FOR THE TREATMENT OF AIDS

SPECIFICATION

FIELD OF THE INVENTION

This invention relates to antiviral agents which inter¬ fere with viral intracellular multiplication.

BACKGROUND OF THE INVENTION

The progressive spreading of the AIDS infection with its tragic consequences on the world population has mobilized worldwide scientific efforts to contain the epidemic and search for effective means to alter the HIV virus life cycle.

The experience acquired in the past with other human viral diseases has immediately suggested that vaccines against the virus be considered and implemented. To this extent, either biosynthetic or extractive fragments of viral glycoprotein envelope, incorporated into appropriate biolog¬ ical carriers, have been injected into animals to assess whether they would be immunogenic (that is capable of causing production of antibodies) and whether the antibodies produced would interact with the virus and prevent its binding to the cells and receptors. Hopefully such an interaction would ameliorate the conditions of the affected subjects and would protect from infection those newly exposed to the virus. As of now, these efforts have failed with respect to the HIV virus since the antibodies, when produced, have not been effective in arresting the viral spread nor the progression of the disease. This ineffectiveness may be caused, at least in part, by the high rate of mutation of the virus, with corresponding changes in the structure of all its gene prod¬ ucts, including the envelope glycoproteins. Thus, a mutated virus may not be recognized by antibodies elicited by a slightly different antigen derived from a previous mutation.

The same arguments may apply to antibodies against the viral reverse transcriptase. This enzyme is responsible for the transcription of the viral genome - RNA - into the (cor¬ responding) DNA which may initiate progeny formation or be incorporated in the genome of the host cell. Moreover, the difficulty of these antibodies to reach the intracellular compartments where the reverse transcriptase is active make their potential usefulness rather questionable.

Other approaches contemplate (1) the synthesis of those peptide fragments of the T-4 viral envelope glycoproteins which engage with the cell receptors prior to internalization of the whole virus; (2) the production of antibodies to the cell viral receptors; and (3) the biosynthesis of soluble fragments of T-4 cell viral receptors (CD4 antigen) capable of intercepting free, extracellular virus and binding it to prevent subsequent internalization. The first two products, by binding and occupying the viral receptors of the sensitive cells would make them unavailable for binding the virus. Considering the variety and the number of cells sensitive to viral attack present in a given organism, and the number of their viral receptors, one wonders whether those peptides or antibodies could be made available with a half-life and in amounts sufficient to keep the viral receptors constantly occupied for a finite amount of time. Yet, approaches to interfere with the binding of the virus to the surface receptors of the sensitive cells are sound because, if successful, they would prevent the inter¬ nalization of the virus which would remain exposed to the other humoral and cellular mechanisms of defense of the organism and would be prevented from incorporating its genome into that of the host cell.

However, such interference should be a permanent and complete one if it were to prevent healthy cells from becom¬ ing infected either from endogenous virus released by dying cells or exogenous virus acquired through the usual means of transmission. An inexpensive, easily available, low molecu¬ lar weight, non-protein compound, capable of diffusing throughout the organism and capable of binding the virus or the viral receptors would be more suitable to this purpose than bulky peptides or proteins of finite half-life, costly preparation and potential immunogenic properties.

SUMMARY OF THE INVENTION

The present invention provides low molecular wight degra¬ dation products of naturally occurring linear polyanion heparin (glycosaminoglycan, acid mucopolysaccharide) lacking antithrombic and anticoagulant activities (i.e., less than 50 units antithro bin activity) , yet retaining sufficient nega¬ tive charges, as effective agents which interfere with the replication of the human immunodeficiency virus (HIV) . The methods of invention provide for inhibiting T cell death and replication of human immunodeficiency virus (HIV) comprising exposing the human T cells to a pharmaceutically effective amount of a low molecular weight heparin degradation products lacking anticoagulant activity. The present invention fur¬ ther provides a method of treating HIV infection by adminis¬ tering to the infected host a therapeutically effective amount of a low molecular weight heparin degradation product lacking anticoagulant activity. The administration can be accomplished through any of many routes including intrave¬ nously, intraperitoneally, orally, subcutaneously, intramus¬ cularly, rectally, topically or by inhalation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Heparin and Its Degradation Products.

Commercial heparin is prepared from beef lung or, prefer¬ entially from bovine or porcine intestinal mucosa by extrac¬ tion procedures based on the high negative charge of mole¬ cule. After digestion of the crude material with proteases, the clear supernatant obtained by centrifugation is treated with quaternary ammonium salts. This precipitation may be achieved step-wise, at decreasing salt concentrations, the most highly charged molecules being obtained as a precipitate at the high salt concentration.

This higher charged heparin may be precipitated with quaternary ammonium salts above 0.35 M NaCl, while the other contaminating glycosaminglycans (chondroitin sulfates, heparan sulfate) remain in solution, to be precipitated upon further dilution of the solution.

The heparin-quaternary ammonium salt precipitate is repeatedly washed in the centrifuge with 95% ethanol/10% potassium acetate in order to remove the quaternary ammonium salt. The potassium salt of heparin is dissolved in water, cleared by centrifugation and further purified by column chromatography on anion or cation exchange resins.

Several methods are available for the isolation of hepa¬ rin fractions of different molecular weight: a) anion ex¬ change chromatography in which the heparin is retained on the column at low ionic strength and fractions of increasing molecular weight may be eluted by step-wise increase in the ionic strengths of the eluting solution (from 0.2 M to 3.0 M NaCl) ; or b) gel filtration, in which larger fractions emerge first from the column, to be followed by gradually smaller ones. In this technique, the influence of sulfate content may be minimized by using as eluant a solution of 0.2 M NaCl.

The relevance of these techniques to the present inven¬ tion depends on the linear correlation between molecular size and anticoagulant activity of heparin. We are particularly interested in heparin fractions lacking anticoagulant activi¬ ty, yet still capable of interfering with the multiplication of the virus and still possessing the properties of stimulat¬ ing cellular endogenous mechanisms of defense. Other method¬ ologies that may be used for the same purpose include affini¬ ty chromatography of heparin on antithrombin-sepharose col¬ umns and factor Xa-sepharose columns in order to separate the high affinity fraction, having very high antithrombic or anticoagulant activity, from the low - affinity ones, essen¬ tially devoid of such activities. Thus, with these tech¬ niques, we were able to screen large and small molecular weight fractions of heparin without dangerous anticoagulant activity, for any possible inhibitory activity on the multi¬ plication of the virus.

Additional chemical and enzymic methods may be used to degrade large molecular weight heparin fractions without further affecting their sulfate content. Dietrick, C.P., Biochem. J. 108: 647-654 (1968). Low molecular weight heparin (from porcine intestinal mucosa) is produced by ni¬ trous acid degradation of native heparin, to a 4,000-6,000 MW. It is obtainable commercially through Sigma Company. This compound still possesses good anti-Xa activity, (> 150 IU/mg) but lower antithrombic activity (>40 IU/mg) than the undegraded original material. This low molecular weight heparin can be completely de-N-sulfated and N-acetylated as described below, in order to obtain a product without anti¬ coagulant activity. Heparin and heparan sulfate are glycosminoglycans which uniquely contain N-sulfate groups. Older methods of de-N- sulfation with controlled hydrolysis in dilute acids (0.04N HC1 at 56° for 4 hours) caused hydrolysis of N-sulfated groups accompanied by some cleavage of glycosidic linkages (depolymerization) and of 0-sulfate groups (de-O-sulfation) . More recently, it has been reported that the pyridinium salt of N-sulfated glucosamine residues (present in heparin and heparan sulfate) is desulfated much more rapidly and selec¬ tively in dimethyl sulfoxide containing small amounts of water or methanol. The sodium salt or heparin (molecular weight 12,00 to 15,000) is dissolved in 25 ml of water and the solution is passed through a column of Dowex 50x8 at 4° C. The effluent and washing are combined, neutralized with pyridine and lyophilized to give the pyridinium salt of heparin.

This salt is dissolved in 100 ml of dimethyl sulfoxide containing 5% water and the solution is kept for 90 minutes at 50^* C, then diluted with equal volume of water. The pH of the solution is adjusted to 9.0-9.5 with 0.1 M NaOH, dialyzed over night against tap water and then against distilled water for 20 hours. Filtration and lyophilization of the retentate gives the sodium salt of completely de-N-sulfated heparin. This large molecular weight compound (12,000 to 15,000 daltons) has no anticoagulant activity. This compound may be acetylated by the method of Danishefsky, et al. Arch. Biochem. , Biophys. 90:114 (1960). 300 micrograms of the sodium salt of completely de-N-sulfated heparin in 30 ml of water are added to 5 ml of methanol and 0.9 ml of acetic anhydride. The mixture is kept for two hours at pH 6.5 by adding 10% NaHC0₃, under stirring at 3-4° C. The reaction mixture is dialyzed against running tap water over night and then against distilled water for 20 hours. Filtration and lyophilization of the retentate gives the sodium salt of completely de-N-sulfated, N-acetylated heparin. If ¹⁴C, ³H or deuterium-labeled acetic anhydride is used, the final acety- lated product will be conveniently labeled. Such a radioac- tively labeled derivative may be useful in following the fate of large molecular weight heparin added to a cell culture or administered to experimental animals.

Another facile and fruitful approach to the preparation of non anticoagulant derivatives of heparin is provided by E. Holmes et al., Hemostasis 16, Supplement 2: 1-7 (1986). Antithro bin (commercially available) bound to an insoluble matrix (Sepharose) may be used to separate large MW heparin into two fractions: one with high and one with low affinity for antithrombin (HA and LA respectively) . The first has approximately 300 U anticoagulant activity/mg; the second is essentially inactive. This technique has allowed to identify the structure of the pentasaccharide of heparin responsible for binding to antithrombin. The internal glucosamine unit carries a 0-sulfate group in position C-3, previously consid¬ ered essential for interaction with antithrombin and absent in LA heparin. More recently, however, the consensus is that the 3-O-sulfate is an artifact produced during deamination of the 2-N-sulfate group, which migrates to position 3. In fact, the 3-O-sulfate group is never found in products of bacterial degradation of heparin. However, three additional sulfate groups, are required for high affinity to antithrom¬ bin. One of those, 2-N sulfate on the internal glucosamine, may be removed by solvolytic cleavage with methyl sulfoxide, leaving a highly sulfated, nonanticoagulant product.

Both these fractions (HA and LA) may be depolymerized with nitrous acid, to obtain fragments of various length which may be separated by gel chromatography. It is well documented that anticoagulant activity of heparin is highly dependent on molecular weight. Thus, oligosaccharides of less than 18 monosaccharides (less than 5,000 MW) length have lost their anticoagulant activity but retained their anti¬ thrombic activity (anti-Xa activity) .

Following the procedures of Pejlaer, G. et al., J. Biol. Chem. 263:5197 (1988), 42 micrograms of sodium nitrate (NaN0₂) are added to one microgram of heparin in 20 ml of ice-cold H₂0, acidified to pH 1.4 with dilute H₂S0₄. After three hours in the ice bath, the oligosaccharides are pre¬ cipitated with 10 volumes ethanol and separated by gel chro¬ matography on sephadex G-50. Fractions composed of 2 to 20 monosaccharides may thus be obtained. The nitrous acid treatment causes an oxidative deamination of N-sulfated glucosamine, with cleavage of the glucosaminidic linkage. Thus, N-sulfated glucosamine is transformed into 2,5- anhydromannose. This residue, located at the reducing end of the oligosaccharide products, may be conveniently reduced with sodium borohydride labeled with tritium (NaB³H₄) , to yield tritium-labeled mannose at the reducing end. This labeling technique allows one to follow the fate of these oligosaccharides¹, once added to a cell culture.

Finally, another method for degrading heparin to di and trisaccharides makes use of the enzyme heparinase II, ob¬ tained originally from Flavobacterium heparinum and now commercially available. 200 mg of heparin in 0.005 M phos¬ phate buffer pH 7.0, containing 0.02% bovine serum albumin, is incubated at 25° C for up to 125 hours with heparinase II (10 units in 10 ml buffer) . A series of disaccharides is obtained, which may be separated by high pressure liquid chromatography. Shorter periods of incubation, with the enzyme, may yield trisaccharides or oligosaccharides which may be separated by gel chromatography. All these products

¹ A note of caution: oligosaccharides prepared by nitrous acid oxidation have been used to produce monoclonal antibodies in mice. Thus, the possible antigenicity of 2,5 anhydro annose must be kept in mind. Pejlaer et al. J. Biol. Chem. 263:5197 (1988) . of enzymic cleavage have a 4-5 unsaturated uronic acid at the non-reducing end and have a MW of about 1,000 to about 3,000 daltons.

Heparin Sample B

Sample B used in these experiments is a degradation product of heparin (MW 4.000-5,000 daltons) prepared by mild oxidation of heparin with copper, H₂02 and ascorbate.

100 g of sodium heparin were dissolved in 10 liters of a solution containing 0.5M NaCl; 0.35M sodium acetate; 0.35% ascorbic acid; 0.45% copper acetate and H₂0₂ to a final concentration of 1.8 volumes. The pH of the solution was adjusted to 7.80 before the addition of peroxide and again immediately after the addition of peroxide.

The solution was incubated at 50^* C, with stirring for 20 hours, samples of 5-7 ml were collected at regular inter¬ vals. The buffered solution, prior to addition of peroxide and final pH adjustment, was pale blue-green in color. Upon addition of peroxide and alkali to pH 7.80, vigorous bubbling ensued, with a raise in the solution temperature to 65° C. The bubbling lasted for 30 minutes after which the tempera¬ ture of the solution slowly returned to 50° C.

At the end of the incubation, the solution was clear and blue-green in color. The samples collected at various times during incubation and a final sample were processed in paral¬ lel throughout the following steps:

1. Passage through 7x1 cm columns of cation ex¬ change resin Dowex 50x4, 200 mesh in the hydrogen form in order to retain the copper and allow recovery of free heparin in the effluent. Its elution was followed simply by monitor¬ ing the strong acidity of the effluent. Whenever the latter became neutral, the elution of heparin was complete. 2. Immediate neutralization of each heparin ali¬ quot with sodium hydroxide, in order to obtain the sodium salt of the glycan.

3. The various aliquots were reduced in volume (if needed) , with a rotary evaporator, under vacuum, and/or directly precipitated in the centrifuge with 2 volumes of absolute methanol. After washing twice with absolute metha¬ nol, each precipitate was washed and dried with ether to the powder state. The material obtained at the end of the incu¬ bation was weighed in order to calculate the yield of the process (80-85%) .

4. Each sample collected during and at the end of the incubation was analyzed by electrophoresis on 6% acryla- mide gels, to verify whether degradation had occurred. Sam¬ ples of original and degraded heparin or Fragmin (a trade- marked product of Kabi Vitrum) and of known products of heparin degradation were run as standards. The electropho¬ resis was run with 0.6M barbital buffer pH 8.60 for a period of one hour. Thereafter, the gels were stained with 0.1% Toluidine Blue in 1% acetic acid in water for 30 minutes. Destaining was carried out in 1% acetic acid, with mild agitation. Destained and dried gels were photographed or scanned to obtain a graphic representation of the degree of degradation.

5. When a final sample showed signs of great heterogeneity, with bands or peak indicating degradation products of different sizes, a water solution of the sample was passed through an appropriate size column of anion ex¬ change resin Dowex 1x2, 100-200 mesh, in the chloride form in order to retain the heparin. This was eluted in discreet fraction of increasing molecular size using increasing con¬ centration of NaCl: from 1.0M to 2.0,3.0 and 4.0M NaCl.

Because this techniques eluted the sodium salt of hepa¬ rin, monitoring the effluent acidity was not feasible. Thus, either one has to monitor the absorption of effluent at 310nm or spot a sample of effluent on filter paper impregnated with alcian blue in order to detect the methachromatic reaction of heparin.

The desired sample was then desalted, by gel filtration and precipitated with methanol and dried with ether as de¬ scribed in 3.

While in this series of experiments copper acetate was used as a source of metal catalyst, iron salts, copper or iron turning may be equally used for the production of OH free radicals (Fentόn reaction) .

Experimental

What follows is a description of the assays performed to demonstrate anti reverse transcriptase activity, if any, of

native heparin (Sample A) and Cu"^""/H₂0₂/ascorbate degraded heparin (Sample B) .

HIV RT ASSAY

lOx Buffer:

1M Tris-HCL, pH 7.4-8.0

1M DTT

1M MgCl₂

1M KC1

10% Triton X-100

6mM GSH

100 ul

Template: lOmM Tris + 0.1 mM EDTA 180 ul 0.5mg/ml rAdT 120 ul Add H₂0 100 ul

400 ul^* (Dilute 1:2 Tris:EDTA)

RXN Mixture (1:5) (1:2) lOx Buffer [H³]-TTP:H₂0 Template Sample^*

35.0 ul 50 ul ^*Supernate of fluid plus cells centrifuged at 4°C.

1. Incubate RXN Mixture 2 hours at 41° C.

2. Spot 20 ul on DE81 strips (handle DE81 paper with gloves on only) .

3. Wash with 5% Na₂HP0₄, 4-6x - 4 min/wash double distilled H₂0 2x - 1 min/wash

95% EtOH 2x - 1 min/wash

4. Place in scintillation vials and Aquasol. Example I

The virus, a source of reverse transcriptase, was used at three, decreasing concentrations, 1.0; 0.3; 0.1. Concentra¬ tion 1 is arbitrary and it is defined, for the purpose of this assay, as that amount of virus which provides sufficient enzyme to cause incorporation of tritiated thymidine triphos- phate (3_HTTP) into the template to the extent of 40,000 cpm. The two heparin preparations A and B have been used at sever¬ al, decreasing concentrations: 1.0; 0.3; 0.1; 0.03 mg/ml.

The data indicate that both preparations cause inhibition of reverse transcriptase, preparation A better than B, with decreasing inhibition at decreasing concentrations. Virus dilution does not seem to enhance inhibitory activity.

Example II

This set of experiments investigated whether the two heparin preparations (A or B) interfere with the multiplica¬ tion of HIV in T cells. Both heparin preparations were added to the cells either 1 hour before the virus was added or 1 hour after the virus was added. The rationale for this is that we did not know whether heparin might gain access to the cell interior by itself or whether it needs the virus to proceed - piggy-back - therein. (See, "Animal Viruses Pro¬ mote the Entry of Polysaccharides with Antiviral Activity into Cells," E. Gonzalez E. L. Carrasco. Biochem. Biophvs. Res. Commun. 146; 1303-1310, 1987) .

In these experiments T cells derived from human adult lymphocytic leukemia (ALL) , line C8166, were used at a concentration of 2 x 10⁵cells in 0.5 ml of medium RPMI1640 (standard medium for such experiments) . This amount of cells was place in each well of a multi-well plate. 0.1 ml of HIV virus suspension (HIV pp213, stock of UTMB Galveston) was added to each well and incubation of the plate was carried out at 37° C for up to 21 days.

The concentration of the virus (1:10 dilution from the stock solution) was adopted so as not to kill the cells immediately but nevertheless to provide total infection of the cells within 4-5 days.

In a first set of experiments, cells were exposed to the heparin preparations A or B by adding the heparin sample to the cells 1 hour before or 1 hour after challenging with virus.

Wells were examined every 2 days and samples from wells were removed to measure cellular presence of a viral p-24 core protein antigen. Cells from these samples were spotted in the microscopic fluorescence slides (Cell Line Associates, Inc. 33 Cargo line, Newfield, NJ, fluorescence slides, 8 wells, 8mm) , and fixed with cold acetone and treated with a fluorescent monoclonal antibody against P24. The fluorescent cells in each sample were counted and expressed as a percent¬ age of all cells, using a fluorescence microscope.

TABLE 1: HEPARIN ADDED 1 HOUR BEFORE VIRUS

Concentrations/ml

HEPARIN A

No Heparin Imcr 0.3mcr O.lmg 0.03mσ

DAY 4 30% pos neg neg neg 1-2% pos

DAY 6 100% pos neg neg neg 1-3% pos

HEPARIN B

DAY 4 30% pos neg neg neg neg

DAY 6 100% pos neg neg neg neg TABLE 2: HEPARIN ADDED 1 HOUR AFTER VIRUS ADDITION

n lmg 0.3mg O.lmg 0.03mg neg neg neg 1-3% pos neg neg neg 1-3% pos

neg neg neg neg

neg neg neg neg

CONTROLS: Cells without virus, all negative.

Cells without virus but with heparin A or B at stated concentrations, to investigate for toxicity, as per visual morphological inspection, revealed that Sample A showed +/- toxicity at lmg concentration only, while Sample B did not show toxicity at any concentration.

EXAMPLE II

An additional experiment was performed with Heparin B, to establish any effect on late infection. The test was designed essentially along the lines described in EXAMPLE II. The same cell line, same HIV virus strain, same fluorescence technique, were used to detect P-24 antigen on cell surface. The differences included: 10 the viral inoculum was carried out with full strength virus concentration, in order to obtain in the controls the full effect of the infection in a short period of time; and 2) the Heparin B was added 48 hours later. The virus killing effect was measured by the tripan blue exclusion method. The rest of the experiment was the same. Wells were examined every day for morphological evi¬ dence of cytopathic effects (CPE) (*) and cell samples were obtained every day for fluorescence microscopy to identify presence of P-24 on cell surface (#) . Heparin B was used at l.Omg; 0.3mg; O.lmg and 0.03mg/ml, as previously noted. Both the cytopathic effects and the amount of fluorescence were scored from 1+ to 4+.

TABLE 3: CPE (*=0) AND FLUORESCENCE (#=0)

Days of 0 1 2 3 4 5 6 7 1 experiment

Control cells, not infected *

# # # # # # # # # # #

Control cells, + Heparin B * for each of #

1.0; 0.3; 01.; and 0.03mg/ml

Control cells infected * 1+ 3+ 3+

# 3+ 3+ 3+ 4+

Cells infected + Heparin B for each of * * * * * * * 1.0;; 0.3; # # 3+ 3+ 3+ 3+ 3+ 0.1; 0.03mg/ml

It is clear that Heparin B protects the cells from being killed by the virus even though they had been infected for 48 hours. Control cells, infected but not treated show morphological changes and fluorescence by day 2 and 3, with cell death by day 5.

Control cells plus Heparin B do not show changes of toxicity. However, cells infected at time 0 and treated 48 hours later do not show cytopathic effects, even though they display the presence of viral core protein (P-24) .

These data indicate that Heparin B, administered 48 hours after viral infection does not prevent the synthesis of the core P-24 antigen of the virus. The latter apparently must have had time to shed the glycoprotein coat, to free its RNA from the core protein, to have the RNA transcripted into DNA and this incorporated into host DNA.

The presence on the cell of P-24 antigen clearly indi¬ cates that the virus has undergone a good part of its intra¬ cellular life cycle. Yet, the treated cells do not show signs of suffering, and survive well. Thus, Heparin B apparently acts quite late in the life cycle of the virus, protecting cells from virus killing.

While the presently preferred embodiments of the inven¬ tion have been set forth for the purposes of disclosure, changes and modifications therein can be made which are within the spirit of the invention as defined by the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of inhibiting the multiplication of HIV in human T cells comprising exposing the human T cells to a pharmaceutically effective amount of low molecular weight heparin degradation product lacking anticoagulant activity, said heparin degradation product obtained by oxidation of heparin using free radical OH.

2. The method of Claim 1 wherein the heparin degrada¬ tion product has a molecular weight ranging from about 4,000 to about 6,000 daltons.

3. The method of claim 1 wherein the heparin degrada¬ tion product is prepared by mild oxidation of heparin using H₂0₂ and a metal catalyst.

4. The method of claim 1 wherein the pharmaceutically effective amount ranges from about lmg/ml to about 0.03 mg/ml.

5. A method of prolonging the survival time of human T cells challenged with the human immunodeficiency virus (HIV) comprising exposing the human T cells to a pharmaceutically effective amount of low molecular weight heparin degradation product lacking anticoagulant activity, said heparin degrada¬ tion product obtained by oxidation of heparin using free radical OH.

6. The method of claim 5 wherein the heparin degrada¬ tion product has a molecular weight ranging from about 4,000 to about 6,000 daltons.

7. The method of claim 5 wherein the heparin degrada¬ tion product is prepared by mild oxidation of heparin.

8. The method of claim 5 wherein the pharmaceutically effective amount ranges from about lmg/ml to about 0.03mg/ml.

9. A method of treating a host infected with human immunodeficiency virus (HIV) comprising administering to the host a pharmaceutically effective amount of a low molecular weight heparin degradation product lacking anticoagulant activity, said heparin degradation product obtained by oxida¬ tion of heparin using free radical OH.

10. The method of claim 9 wherein the heparin degrada¬ tion product has a molecular weight ranging from about 4,000 to about 6,000 daltons.

11. The method of claim 9 wherein the heparin degrada¬ tion product is prepared by mild oxidation of heparin.

12. The method of claim 9 wherein the pharmaceutically effective amount ranges from about lmg/ml to about 0.03mg/ml.

13. The method of claim 9 wherein the heparin degrada¬ tion product is administered orally, intravenously, or intraperitoneally, subcutaneously, intramuscularly, topical¬ ly, rectally or by inhalation.

14. A pharmaceutical composition for treating human immunodeficiency virus (HIV) infection comprising: a pharmaceutically effective amount of a low molecular weight heparin degradation product lacking anticoagulant activity together with a pharmaceutically acceptable diluent, said heparin degradation product obtained by oxida¬ tion of heparin using free radical OH.

15. The pharmaceutical composition of claim 14 wherein the heparin degradation product has a molecular weight rang¬ ing from about 4,000 to about 6,000 daltons.

16. The pharmaceutical composition of claim 14 wherein the heparin degradation product is prepared by mild oxidation of heparin.

17. The pharmaceutical composition of claim 14 wherein the pharmaceutically effective amount ranges from about lmg/ml to about 0.03mg/ml.